Ohr plays a central role in bacterial responses against PNAS PLUS fatty acid hydroperoxides and peroxynitrite

Thiago G. P. Alegriaa,1, Diogo A. Meirelesa,1, José R. R. Cussiola,2, Martín Hugob,3, Madia Trujillob, Marcos Antonio de Oliveirac, Sayuri Miyamotod, Raphael F. Queirozd,4, Napoleão Fonseca Valadarese,5, Richard C. Garratte, Rafael Radib,6, Paolo Di Masciod, Ohara Augustod, and Luis E. S. Nettoa,6

aDepartamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, 05508-090, Sao Paulo, Brazil; bDepartamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay; cDepartamento de Biologia, Universidade Estadual Paulista Júlio de Mesquita Filho, Campus do Litoral Paulista, 11330-900, Sao Vicente, Brazil; dDepartamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-000, Sao Paulo, Brazil; andeInstituto de Física de São Carlos, Universidade de São Paulo, 13566-570, Sao Carlos, Brazil

Contributed by Rafael Radi, November 30, 2016 (sent for review October 8, 2016; reviewed by Leopold Flohé and Leslie B. Poole) Organic hydroperoxide resistance (Ohr) are unique Cys- Prx and Gpx. Ohr was first described in Xanthomonas campestris, based, lipoyl-dependent . Here, we investigated the through the characterization of a null mutant strain (Δohr). The involvement of Ohr in bacterial responses toward distinct hydroper- Δohr strain displays high sensitivity toward tert-butyl hydroper- oxides. In silico results indicated that fatty acid (but not cholesterol) oxide (t-BHP), but not to H2O2. Moreover, transcription of the hydroperoxides docked well into the active site of Ohr from Xylella ohr is specifically induced by t-BHP (14). Later on, this fastidiosa and were efficiently reduced by the recombinant enzyme was found in several bacteria (15). Subsequently, we and – as assessed by a lipoamide-lipoamide dehydrogenase coupled assay. others were able to show that this “organic hydroperoxide re- Indeed, the rate constants between Ohr and several fatty acid hydro- − − sistance” phenotype is due to the Cys-based, thiol-dependent peroxides were in the 107–108 M 1·s 1 range as determined by a competition assay developed here. Reduction of peroxynitrite by activity of Ohr (16, 17). In contrast, OsmC Ohr was also determined to be in the order of 107 M−1·s−1 at pH 7.4 were initially implicated in the osmotic stress response (18), and only later were shown to share structural and biochemical simi-

through two independent competition assays. A similar trend was BIOCHEMISTRY observed when studying the sensitivities of a Δohr mutant of Pseu- larities with Ohr (19, 20). Ohr/OsmC enzymes present an domonas aeruginosa toward different hydroperoxides. Fatty acid hy- α/β-fold that is very distinct from the thioredoxin-fold charac- droperoxides, which are readily solubilized by bacterial surfactants, teristic of Prx and Gpx (17, 21, 22). Furthermore, Ohr/OsmC killed the Δohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for and glutathione peroxi- Significance dases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because Hydroperoxides play central roles in cell signaling. Hydroperoxides Δ the ohr mutant was more sensitive than wild type to 3-morpholi- of arachidonic acid are mediators of inflammatory processes in nosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In mammals, whereas hydroperoxides of linoleic acid play equivalent the case of H2O2 insult, cells treated with 3-amino-1,2,4-triazole roles in plants. Peroxynitrite is also involved in host–pathogen (a inhibitor) were the most sensitive. Furthermore, fatty acid interactions, and hydroperoxide levels must therefore be strictly hydroperoxide and SIN-1 both induced Ohr expression in the wild- controlled by host-derived thiol-dependent peroxidases. Organic type strain. In conclusion, Ohr plays a central role in modulating the hydroperoxide resistance (Ohr) enzymes, which are present in levels of fatty acid hydroperoxides and peroxynitrite, both of which many bacteria, display unique biochemical properties, reducing – are involved in host pathogen interactions. fatty acid hydroperoxides and peroxynitrite with extraordinary efficiency. Furthermore, Ohr (but not other thiol-dependent per- hydroperoxides | thiols | Cys-based peroxidase | pathogenic bacteria | oxidases) is involved in the Pseudomonas aeruginosa response to Pseudomonas aeruginosa fatty acid hydroperoxides and to peroxynitrite, although the latter is more complex, probably depending on other enzymes. There- hiol peroxidases are receiving increasing attention as biological fore, Ohr plays central roles in the bacterial response to two hy- Tsensors and signal transducers of hydroperoxide-dependent droperoxides that are at the host–pathogen interface. pathways (1–3). (Prx) and (Gpx) are central players in hydroperoxide metabolism and sens- Author contributions: T.G.P.A., D.A.M., J.R.R.C., M.H., M.T., S.M., R.R., P.D.M., O.A., and ing, which is consistent with their high overall abundance and re- L.E.S.N. designed research; T.G.P.A., D.A.M., J.R.R.C., M.T., M.A.d.O., S.M., R.F.Q., and N.F.V. performed research; M.H. and M.T. contributed new reagents/analytic tools; T.G.P.A., D.A.M., activity (3). J.R.R.C., M.T., R.C.G., R.R., O.A., and L.E.S.N. analyzed data; and T.G.P.A., D.A.M., M.T., R.C.G., The issue of substrate specificity in Cys-based peroxidases is R.R., and L.E.S.N. wrote the paper. intriguing. Prx enzymes are highly reactive to hydroperoxides but Reviewers: L.F., University of Padua; and L.B.P., Wake Forest School of Medicine. not to other oxidants or thiol reagents, such as chloroamines or The authors declare no conflict of interest. iodoacetamide (4). The structural features responsible for this 1T.G.P.A. and D.A.M contributed equally to this work. phenomenon are still emerging (5–7), including some more subtle 2Present address: Departamento de Bioquímica, Instituto de Química, Universidade de aspects of their specificity. For instance, the three different Prxs Sao Paulo, 05508-000, Sao Paulo, Brazil. from Mycobacterium tuberculosis display distinct hydroperoxide 3Present address: German Institute of Human Nutrition, 14558 Potsdam, Germany. specificities with alkyl hydroperoxide reductase E (AhpE) being 4Present address: Departamento de Ciências Naturais, Universidade Estadual do Sudoeste highly specialized toward long-chain fatty acid hydroperoxides (8). da Bahia, Vitória da Conquista, 45083-900, Bahia, Brazil. Furthermore, yeast Tsa1, yeast Tsa2, rat Prx6, and human Prx2 are 5 – Present address: Laboratório de Biofísica Molecular, Departamento de Biologia Celular, more reactive toward H2O2 (9 11), whereas human Prx5 is more Universidade de Brasília, CEP 70910-900, Brasília, Brazil. reactive toward organic hydroperoxides and peroxynitrite (12, 13). 6To whom correspondence may be addressed. Email: [email protected] or nettoles@ib. Organic hydroperoxide resistance (Ohr) and osmotically in- usp.br. ducible C (OsmC) form another group of Cys-based This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. peroxidases that are biochemically and structurally distinct from 1073/pnas.1619659114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1619659114 PNAS Early Edition | 1of10 Downloaded by guest on September 30, 2021 enzymes accept electrons from lipoyl groups of proteins (23, 24) in contrast to Prx and Gpx, which are most commonly reduced by thioredoxin or glutaredoxin/glutathione (GSH) (1, 2). Despite all of these studies, the identification of the natural oxidizing substrates of Ohr is still a challenge. It is known that the Ohr from Xylella fastidiosa (XfOhr) is 10,000-fold more efficient in the removal of t-BHP compared with H2O2 (23). However, be- cause t-BHP and cumene hydroperoxide (CHP) are artificial compounds, the identity of the biological oxidizing substrates for Ohr remains to be identified. We previously raised the hypothesis that elongated hydrophobic molecules, such as hydroperoxides derived from fatty acids, could be the natural substrates, because these compounds fitted very well into the electron density corre- sponding to a PEG molecule found bound to the active site of Ohr in the crystal structure (22). Other corroborating evidence came from the observation that the Ohr and its transcriptional repressor (OhrR) system are responsible for linoleic acid hydroperoxide (LAOOH) clearance in Xanthomonas spp. (25). Furthermore, LAOOHs induce ohr transcription, at higher levels than t-BHP in some cases (25–27). Here, we systematically investigated the involvement of Ohr in the bacterial response toward distinct hydroperoxides. Initially, using different approaches, we showed that XfOhr preferentially reduced long-chain fatty acid hydroperoxides and also perox- ynitrite. Furthermore, microbiological assays using Pseudomonas aeruginosa correlated well with the biochemical/kinetic assays. Taken together, our results indicated that Ohr protein is central to the response of the bacterium to fatty acid hydroperoxides and peroxynitrite, although the process appears to be more complex Fig. 1. Molecular docking of long-chain hydroperoxides into the Ohr active in the latter case, possibly involving the cooperation of other site pocket. The molecular surface of XfOhr (PDB ID code 1ZB8) is repre- sented in brown, whereas the hydroperoxides are shown as lines with the enzymes. In contrast, catalase is a central player in the response = of P. aeruginosa to exogenously added H O . The experimental atoms colored as follows: carbon purple (A and B) and cyan (C and D), 2 2 oxygen = red, and hydrogen = white. (A and C) Results of docking simula- approach presented herein allows defining the relative sensing tions, showing the entire protein structure. Ligands used hydroperoxides and detoxifying roles of various peroxidatic systems in bacteria derived from OAOOH, palmitoleic acid hydroperoxide, and LAOOH (A)and challenged with different biologically relevant hydroperoxides. arachidonic acid hydroperoxide (C): 5-HpETE, 5(S)-HpETE, 8-HpETE, 9-HpETE, 12-HpETE, 15-HpETE, and 15(S)-HpETE. Oleic and linoleic acid molecules Results containing the hydroperoxide group in both the cis- and trans-configura- Fatty Acid Hydroperoxides Dock into the Ohr Active Site. Previous tions or in different combinations of the two (linoleic hydroperoxides) were data suggested that the active site cavity of XfOhr preferentially considered in the analysis. (B and D) Details of the Ohr active site pocket, accommodates elongated molecules (22). Motivated by this ob- showing the hydroperoxide groups of the fatty acid hydroperoxides facing γ 61 servation, we performed an unbiased docking analysis to gain toward the S of Cys . The atoms of the enzyme are represented as space- filling spheres and colored brown, except for the sulfur atom of Cys61 (yellow). further insight into the affinity of Ohr protein for different organic The docking procedures were performed using Gold software (70), and mo- hydroperoxides. The full structure of XfOhr [ lecular graphics were generated using PyMOL (www.pymol.org/). (PDB) ID code 1ZB8] was analyzed against all cis and trans iso- mers of hydroperoxides derived from mono- or polyunsaturated fatty acids (Fig. 1 A and C). tives, the lower fitness values are probably due to the failure to Long-chain fatty acid hydroperoxides docked well into the orient the hydroperoxide moiety adequately toward the catalytic XfOhr active site, overlapping with the position occupied by a cysteine and/or to their rigidity, which prohibits optimizing hydro- PEG molecule in the Ohr crystal structure. All of the elongated phobic interactions within the binding site cavity. These data sup- hydroperoxide molecules produced good fits with their O–O bond port the hypothesis that elongated hydrophobic molecules are the pointing toward the catalytic cysteine (Cys61, so-called peroxidatic preferred oxidizing substrates of Ohr proteins (22). Cys), although trans isomersgavehigherscores(Fig.1B and D). However, it was not possible to observe any preferred orientation Biochemical Characterization of Peroxidase Specificity. Next, we tested of the carboxyl group in relation to the surface of the enzyme, the ability of XfOhr to reduce various lipid-derived hydroperoxides suggesting that hydrophobic interactions are major factors for the to validate the docking analyses. Thiol peroxidase activity was binding and orientation of the hydroperoxide within the active site assessed using the previously described lipoamide-lipoamide de- (Fig. 1 and Fig. S1A). This finding is consistent with the fact that no hydrogenase–coupled assay (23). In contrast to other thiol- systematic difference could be observed between S and R enan- dependent peroxidases, lipoylated proteins (but not thioredoxin or tiomers. In the case of hydroperoxides containing more than one glutathione) are the most probable physiological reductants of peroxidation, no preference for the peroxide group pointing to- XfOhr and osmotically inducible protein C from Escherichia coli ward Cys61 was detected. However, in stark contrast, neither the cis (EcOsmC) (23). However, alternative ways of Ohr reduction (e.g., nor trans isomers of cholesterol hydroperoxide (ChOOH) docked redoxins) cannot be excluded and should be tested in the future. well into the active site cavity (Fig. S1B). For example, the fitness Following the reduction of an artificial organic hydroper- scores for the docked ChOOHs, which varied between 30.1 and oxide (t-BHP) and applying a bisubstrate steady-state approach, it 47.1, were, on average, 38% lower than the fitness scores for hy- was previously determined that Ohr displays a ping-pong kinetic droperoxides derived from arachidonic acid (54.8–67.3). This mechanism, and the true Km values for dihydrolipoamide and lip- finding is all the more significant, given that the cholesterol de- oylated proteins are in the 10–50 μMrange(23). rivatives contain more atoms; therefore, the ligand efficiency for A major challenge here was to establish conditions for solubi- these molecules would be even lower. For the cholesterol deriva- lizing hydrophobic hydroperoxides without compromising the

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1619659114 Alegria et al. Downloaded by guest on September 30, 2021 structure and activity of XfOhr. Initially, XfOhr activity was eval- docking analysis. In these experiments, Triton X-100 (0.15%) was PNAS PLUS uated in the presence of a series of detergents and organic solvents used instead of Tween-20 (0.25%) to solubilize ChOOH, because (Fig. S2A), using a highly water-soluble organic hydroperoxide, this procedure results in good solubilization of this lipid (30). The t-BHP, as a substrate. XfOhr retained more than 80% of its original products of oleic hydroperoxide (OAOOH) and LAOOH re- activity toward t-BHP when Tween-20 (0.25%) was used. At the duction by XfOhr were confirmed to be the corresponding alcohols same time, the activity toward LAOOH was maximal (Fig. S2B), by mass spectrometry (Fig. S3). indicating good solubilization of LAOOH (100 μM). Therefore, Regarding the non-Michaelis–Menten behavior of XfOhr at Tween-20 was chosen to solubilize hydrophobic hydroperoxides in substrate concentrations higher than 20 μM(Fig.2A), two non- the lipoamide-lipoamide dehydrogenase–coupled assays. exclusive hypotheses can be proposed: micelle formation of lipid Reduction of LAOOH by XfOhr presented a complex steady- hydroperoxide molecules and/or hyperoxidation of the peroxidatic 61 − state kinetic behavior that was biphasic: At low concentrations, up cysteine of XfOhr (Cys ) to sulfinic acid (Cys-SO2H ) or sulfonic − to 20 μM, the rate of the reaction increased hyperbolically with acid (Cys-SO3H )(Fig.3A). increasing concentrations of LAOOH, as expected for a Michaelis– Considering the first hypothesis, the critical micelle concentra- Menten model (Fig. 2A). However, above 20 μM, the rate dropped tion (CMC) lies in the range of 6–60 μM for most unsaturated fatty markedly as a function of LAOOH concentration (Fig. 2A). acids, depending on pH and the counter-cation used (31–34), Considering only the first phase (up to 20 μM), we estimated which is consistent with the inflection point of 20 μM(Fig.2A). −1 by nonlinear regression a kcat app of 36 s and a Km app of 7.3 μM However, Tween-20 was present at a 0.25% concentration, which at 50 μM dihydrolipoamide (Fig. 2A, Inset), which indicated that appears to solubilize LAOOH, at least up to a 100-μM concen- XfOhr is highly efficient in the removal of LAOOH (kcat app/Kmapp= tration (Fig. S2B). − − 5 × 106 M 1·s 1). However, one major limitation of this analysis So, we investigated if hyperoxidation could be involved in the was that saturation was not achieved; therefore, the kinetic pa- second phase decay (Fig. 2A). Among Cys-based peroxidases, it is rameters have probably been underestimated. Consistent with this well known that sulfenic acid (Cys-SOH) formed in the peroxidatic result, we note that data for Ohr from Mycobacterium smegmatis Cys can have two fates: (i) disulfide formation through a conden- also did not fit well to the Michaelis–Menten model at high sation reaction toward a second Cys residue or (ii) hyperoxidation LAOOH concentrations; however, in this case, kcat app/Kmappwas tosulfinicacidandsulfonicacidduetoareactionwithasecond − − estimated to be in the range of 105 M 1·s 1 (24). or third hydroperoxide molecule, respectively (Fig. 3A). Because The catalytic efficiency for LAOOH was in the same range as sulfinic acid and sulfonic acid are not reducible by dihy- BIOCHEMISTRY the range previously determined for t-BHP, considering true kcat drolipoamide, such reactions would result in XfOhr inactivation and Km values in the latter case (23). Unfortunately, the biphasic and could explain the rate drop at concentrations above 20 μM shape of the curves (Fig. 2A) does not allow us to obtain true LAOOH (Fig. 2A). enzymatic parameters for fatty acid hydroperoxides. Therefore, We next evaluated the remaining activity of XfOhr after treat- it was not possible to compare the reduction of fatty acid and ment with several hydroperoxides in the presence of Triton X-100 artificial organic hydroperoxides on the same grounds. (Fig. 3B)orTween-20(Fig. S4). In the conditions used, t-BHP and We also evaluated the possibility that XfOhr could reduce hy- CHP (Fig. 3B, c and d) inactivated XfOhr only slightly. In contrast, droperoxides derived from unsaturated fatty acids attached to fatty acid hydroperoxides (OAOOH and LAOOH) inactivated phosphatidylcholine, which could reflect the capacity of the enzyme XfOhr at very low concentrations (Fig. 3B, a and b), whereas H2O2 to reduce hydroperoxides resident in membranes, similar to the and ChOOH did not affect this peroxidase (Fig. 3B, e and f). activity described for phospholipid Gpx4 (28, 29). However, no In parallel, the same samples were also analyzed by non- such activity for XfOhr was detected, although it was capable of reducing SDS/PAGE and the pattern of XfOhr inactivation by reducing both t-BHP and LAOOH in the same experiment (Fig. various hydroperoxides correlated to its oxidation state (Fig. 3B, 2B). Moreover, when phosphatidylcholine hydroperoxide was in- Lower and Fig. S4, Lower). Previously, we have characterized in cubated with phospholipase A2 (PLA2), releasing the correspond- detail by mass spectrometry the XfOhr oxidation states corre- ing free fatty acid hydroperoxide, NADH consumption was sponding to each one of the bands (22). The intramolecular observed (Fig. 2C). Therefore, it appears that fatty acid hydro- disulfide form of XfOhr has a lower hydrodynamic volume, and peroxides attached to phosphatidylcholine were not accessible to consequently runs faster (band A), than the reduced and over- the XfOhr active site. Furthermore, under the same conditions, oxidized forms (band B) of the enzyme (Fig. 3B, Lower and Fig. XfOhr did not reduce ChOOH (Fig. 2B), consistent with the S4, Lower). Hyperoxidation (assessed by appearance of band B)

Fig. 2. Lipoamide-lipoamide dehydrogenase–coupled assay for the reduction of lipid hydroperoxides by XfOhr. Peroxidase activity was monitored by the con- sumption of NADH at 340 nm in the presence of XfOhr (0.05 μM), lipoamide dehydrogenase from X. fastidiosa (XfLpD, 0.5 μM), and lipoamide (50 μM) in sodium phosphate buffer (20 mM, pH 7.4) and DTPA (1 mM). (A) Dose-dependent reduction of LAOOH by XfOhr in the presence of Tween-20 (0.25%). (Inset)Plotofthe rates of NADH oxidation, considering points up to 20-μM concentration of LAOOH. (B) Reduction of hydroperoxides (100 μM) in the presence of Triton X-100

(0.15%). PCOOH, phosphatidylcholine hydroperoxide. (C) Peroxidase activity of Ohr on PCOOH, measured after indicated time of treatment with PLA2.

Alegria et al. PNAS Early Edition | 3of10 Downloaded by guest on September 30, 2021 Fig. 3. Inactivation/hyperoxidation of Ohr. (A) Schematic catalytic cycle of Ohr and its hyperoxidation: Reaction of reduced Ohr with hydroperoxides generates

the peroxidatic Cys (Cysp)-SOH intermediate, which undergo condensation with resolution Cys (CysR = Cys125). This intramolecular disulfide runs faster than the reduced form in nonreducing SDS/PAGE (22). The intramolecular disulfide can be reduced back by dihydrolipoamide. In the presence of high amounts of organic − − hydroperoxides, Cysp-SOH can be further oxidized to Cysp-SO2H (sulfinic acid) or Cysp-SO3H (sulfonic acid), which comigrates with the reduced form of Ohr in − − SDS/PAGE. Cysp-SO2H or Cysp-SO3H cannot be reduced by dihydrolipoamide. (B) Oxidative inactivation/hyperoxidation of XfOhr by various hydroperoxides in the presence of 0.1% Triton X-100. Reduced XfOhr (10 μM) was incubated for 1 h at 37 °C with distinct hydroperoxides at the indicated concentrations: OAOOH

(a), LAOOH (b), t-BHP (c), CHP (d), ChOOH (e), and H2O2 (f). Main panels illustrate residual activity measured by the lipoamide-lipoamide dehydrogenase–coupled assay, using 200 μM t-BHP as a substrate, in the presence of 0.05 μM XfOhr, 0.5 μM lipoamide dehydrogenase from X. fastidiosa (XfLpD), and 50 μM dihydrolipoamide. Each point represents an average of triplicates, and the respective bars correspond to the SD. (Insets) Nonreducing SDS/PAGE, with each well corresponding to an aliquot of Ohr incubated with different concentrations of hydroperoxides (0, 10, 20, 50, and 100 μM).Ineachwell,2μg of total protein was applied.

could only be observed in conditions where some inactivation XfOhr active site, because this molecule can form micelles. Indeed, was observed (Fig. 3B and Fig. S4). the parental compound (cholesterol) presents very a low CMC Inadvertently, Triton X-100 (Fig. 3B, Lower) or Tween-20 (25–40 nM) (36). On the other hand, because Triton X-100 and (Fig. S4, Lower) provoked oxidation of XfOhr to the intra- Tween-20 at least partly solubilize ChOOH (30), our results (Fig. molecular disulfide form, even with no addition of hydroperox- 3B, Lower and Fig. S4, Lower) suggested that this hydroperoxide is ide. This oxidation might be related to the partial structural loss not a good substrate for XfOhr. Also, H2O2 did not inactivate or of XfOhr due to the detergent treatment (Fig. 2A, Inset and Fig. hyperoxidize XfOhr in the analyzed conditions (Fig. 3B), consistent S2). Another possible explanation is that traces of hydroperox- with the low reactivity previously described (22, 23). ides present in detergents could have provoked the oxidation of For other Cys-based peroxidases, substrate specificity corre- XfOhr (35). lates well with hyperoxidation rates (8, 37–39), which is consis- For ChOOH, no evidence of a reaction was obtained. These tent with the fact that oxidation to Cys-SOH and hyperoxidation data agree well with the docking analysis (Fig. 1). However, we are both reactions dependent on hydroperoxide concentration cannot exclude the possibility that ChOOH could not access the (Fig. 3A). Noteworthy, E. coli Tpx also displays biphasic

Table 1. Parameters related to hydroperoxide reduction by Ohr −1 −1 kobs,M ·s Oxidative inactivation, μMMICwt/MICΔohr*

† Hydroperoxide AhpE/Ohr competition ∼IC10 Extent of inhibition

3 H2O2 (3.0 ± 0.6) × 10 >100 1 LAOOH (6.3 ± 1.7) × 107 1 >10 OAOOH (4.5 ± 3.5) × 108 1 >10 5(S)-HpETE (2.6 ± 1.2) × 107 ND ND 15(S)-HpETE (6.0 ± 3.0) × 107 ND ND ChOOH — >100 1 ‡ t-BHP 2.0 × 106 100 3 ONOOH (2 ± 0.3) × 107 ND — CHP ND 100 3

kobs, observed rate constant; ND, not determined. *Ratio of MICwt (minimal concentration of hydroperoxide that inhibits growth of wild-type strain) to MICΔohr (minimal concentration of hydroperoxide that inhibits growth of Δohr strain). † Defined here as the amount of hydroperoxide that provokes 10% Ohr inactivation. ‡ Because Ohr is several orders of magnitude more efficient than MtAhpE in the reduction of t-BHP, it was not possible to determine this rate constant through the competitive assay. Instead, data from the study by Cussiol et al. (23) were added here.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1619659114 Alegria et al. Downloaded by guest on September 30, 2021 3 −1 −1 PNAS PLUS Michaelis–Menten curves with major hyperoxidation by fatty between XfOhr and H2O2 was estimated to be 3.0 ± 0.6 × 10 M ·s . acid hydroperoxides at concentrations above 10 μM, and H2O2 is Once again, XfOhr blocked with NEM lost its capacity to compete again a poor substrate (37). with MtAhpE for H2O2. Previously, by a steady-state approach (the Therefore, considering the amount of hydroperoxide that lipoamide-lipoamide dehydrogenase–coupled assay), we determined provokes 10% Ohr inactivation, we estimated that the reactivity that the catalytic efficiency (kcat/Km)ofXfOhr for H2O2 was (2.3 ± − − of XfOhr to tested compounds increased in the following order: 0.2) × 102 M 1·s 1 (23). H2O2 <<<< CHP < t-BHP < LAOOH ∼ OAOOH (Table 1). This Ohr/AhpE competition assay was performed for several These data are also consistent with the idea that fatty acid hy- hydroperoxides of distinct chemical structures (Fig. S6), and it was droperoxides are the preferred substrates for Ohr proteins. possible to establish the respective rate constants (Table 1). PaOhr was also assessed using this competitive assay, and essentially the Competition and Fast Kinetics Assays on the Oxidation of Ohr. Next, same results were obtained as for the enzyme from X. fastidiosa. we pursued the determination of the rate constants for the re- For instance, the second-order rate constant between OAOOH − − actions between XfOhr and various hydroperoxides. This task is and PaOhr is also in the range of 108 M 1·s 1 (Fig. S6J). not simple because there are not many assays for organic hy- Once again, the same trend was observed: Ohr proteins dis- droperoxides in comparison to the tools available for peroxyni- played higher reactivity toward hydroperoxides with an elon- trite and H2O2. Therefore, we developed a competitive assay gated shape and hydrophobic properties. However, it was not between XfOhr and AhpE from M. tuberculosis (MtAhpE) for possible to determine the rate constant for the reaction between distinct hydroperoxides, monitoring the intrinsic fluorescence of XfOhr and ChOOH because the intrinsic fluorescence of MtAhpE (further details are provided in Materials and Methods) MtAhpE did not change upon treatment with this oxidant. One and taking advantage of the fact that XfOhr and Ohr from P. exception was peroxynitrite, which is described below. aeruginosa (PaOhr) lack Trp residues, and therefore present no such intrinsic fluorescence when excited at 295 nm (Fig. S5A). Reactivity of XfOhr Toward Peroxynitrite. Pathogens can be exposed To validate this AhpE/Ohr competitive assay, we first investi- to peroxynitrite during host–pathogen interactions (40–42). Because gated t-BHP, because the corresponding rate constants are already the reactivity of Ohr proteins toward peroxynitrite has not been known for both Cys-based peroxidases. For instance, the rate hitherto reported, this analysis was performed here. Reduced XfOhr constant for the reaction between t-BHP and MtAhpE is quite low, caused peroxynitrite to decay faster, in a dose-dependent manner − − 8 × 103 M 1·s 1 (8), compared with the corresponding value for (Fig. 5A). The reaction was so rapid that it precluded the determi-

XfOhr and t-BHP, which is two to three orders of magnitude higher nation of the rate constant by this direct approach. Rather, the rate BIOCHEMISTRY (23). As expected, XfOhr strongly inhibited the drop in MtAhpE constant was determined by two independent competitive assays fluorescence induced by t-BHP (Fig. 4A). against HRP (11) or against MtAhpE (as described above). XfOhr at We went on to evaluate the competition between XfOhr and concentrations considerably lower than HRP (10 μM) inhibited the MtAhpE for several organic hydroperoxides. As an illustrative formation of compound I by peroxynitrite (0.4 μM) (Fig. 5B). From example, we show here the competition of the two Cys-based the dose–response effect of XfOhr on peroxynitrite-mediated HRP peroxidases for 15-hydroperoxyeicosatetraenoic acid [15(S)- compound I formation, an apparent rate constant of (1.2 ± 0.4) × − − HpETE], an arachidonic acid hydroperoxide. Reduced XfOhr 107 M 1·s 1 at pH 7.4 and 25 °C was determined (Fig. 5B, Inset). inhibited the decrease of MtAhpE fluorescence caused by 15(S)- As an alternative, we also analyzed the competition between HpETE in a concentration-dependent manner (Fig. 4B), and the MtAhpE and XfOhr for peroxynitrite. Peroxynitrite (0.5 μM) caused second-order rate constant between XfOhr and 15(S)-HpETE a rapid decrease in total fluorescence intensity (λex = 295 nm) of − − was determined to be 6 ± 3 × 107 M 1·s 1. As anticipated, XfOhr reduced MtAhpE (2.5 μM). The exponential fitting of the experi- previously alkylated with N-ethylmaleimide (NEM) completely mental curve was consistent with a rate constant of peroxynitrite- − − lost its ability to inhibit MtAhpE oxidation (Fig. S5B). mediated MtAhpE oxidation of 1.1 × 107 M 1·s 1 at pH 7.4 and A second example is the competition between the two peroxi- 25 °C, in close agreement to the previously reported value (8). Re- dases for H2O2, considering that the rate constant of MtAhpE for duced XfOhr dose-dependently inhibited the decrease in fluores- 4 −1 −1 H2O2 is 8.1 × 10 M ·s (8). We observed that only at very high cence of MtAhpE caused by peroxynitrite, consistent with a rate − − concentrations (10–30 μM) was XfOhr able to inhibit MtAhpE constant between peroxynitrite and XfOhr of (2.0 ± 0.3) ≥ 107 M 1·s 1 (2 μM) oxidation (Fig. 4C). The rate constant of the reaction at pH 7.4 and 25 °C (Fig. 5C). Therefore, XfOhr reduces not only

Fig. 4. AhpE/XfOhr competitive assay for distinct hydroperoxides. Reactions were carried out in potassium phosphate buffer (100 mM) and DTPA (50 μM), pH 7.4, at 25 °C. (A) MtAhpE (2 μM) and t-BHP (1.8 μM) in the presence (red line) or absence (black line) of XfOhr (2 μM). (B) Emission spectrum of MtAhpE (2 μM) in the reduced state (black) and after oxidation by 1.7 μM 15(S)-HpETE (red). To the reaction mixture represented by the red line, XfOhr was added at the following concentrations: 1.0 μM (brown), 2.0 μM (blue), 4.0 μM (green), and 8.0 μM (purple). (C) Emission spectrum of AhpE (2 μM) in the reduced state (black) and after oxidation by 1.8 μM hydrogen peroxide (red). To the reaction mixture represented by red line, XfOhr was added at the following con- centrations: 10.0 μM (blue) and 30.0 μM (green). RFU, relative fluorescence units.

Alegria et al. PNAS Early Edition | 5of10 Downloaded by guest on September 30, 2021 Fig. 5. Peroxynitrite reduction by XfOhr. Reactions were carried out in potassium phosphate buffer (100 mM), DTPA (100 μM), pH 7.4, at 25 °C. (A) Peroxynitrite (50 μM) was rapidly mixed with reduced Ohr at increasing concentrations, and oxidant decay was followed by its intrinsic absorbance at 310 nm. (B) Peroxynitrite (0.4 μM) was mixed with HRP (10 μM) in the absence and presence of increasing concentrations of reduced Ohr. HRP-compound I formation was monitored at the Soret band. The line represents the expected results simulated using the Gepasi program (76), and considering a simple competition between reduced XfOhr and HRP for peroxynitrite, a rate constant of HRP-mediated peroxynitrite reduction of 2.2 × 106 M−1·s−1 and a rate constant of XfOhr-mediated peroxynitrite reduction − − of 1.2 × 107 M 1·s 1, calculated according to Eq. 1.(Inset) Time course of compound I formation by the reaction of HRP (10 μM) with peroxynitrite (0.4 μM) in the absence or presence of increasing concentrations of reduced Ohr. (C) Peroxynitrite (0.5 μM) was mixed with reduced MtAhpE (2.5 μM) in the absence or presence of increasing concentrations of reduced XfOhr, and MtAhpE oxidation was followed by its total intrinsic fluorescence decrease (λ = 295 nm).

fatty acid hydroperoxides but also peroxynitrite, with extraordi- (Fig. 7A). Expression of thiol-dependent peroxidases in the Δohr narily high rates. background was ascertained by Western blot (Fig. S8A). Finally, as expected for a gene central to the bacterial response to fatty In Vivo Characterization of Ohr Sensitivities Toward Various Hydroperoxides. acid hydroperoxides, treatment with LAOOH led to higher levels We aimed to investigate if these results on substrate specificity of ohr induction than did t-BHP (Fig. 7C and Fig. S8B). For correlate to bacterial responses toward distinct hydroperoxides. comparison purposes, we also show here that the expression of For this purpose, we used P. aeruginosa, which produces surfactants the ohr gene in the ΔohrR background was maximal, because the and secretes lipases, allowing it to solubilize and take up very hy- repression by the transcription factor OhrR was absent (Fig. 7C). drophobic compounds (43). In contrast to the LAOOH insult, the response of P. aerugi- The sensitivities of Δohr and wild-type strains were comparatively nosa to exogenous H O was highly dependent on catalase, be- analyzed by the minimal inhibitory concentration (MIC) assay 2 2 against various hydroperoxides. Interestingly, none of the lipid hy- cause increased sensitivity toward the wild-type strain was only droperoxides in the concentration range studied here (OAOOH, detected in cells treated with 3-amino-1,2,4-triazole (ATZ), a LAOOH, and ChOOH) inhibited the growth of the wild-type strain, whereas H2O2, t-BHP,andCHPdid(Fig.6).Asexpected,theΔohr strain presented a reduced capacity to grow in the presence of either artificial (CHP and t-BHP) or fatty acid hydroperoxides compared with the wild type (Fig. 6). It is noteworthy that the extent of in- hibition (defined here as MICwild-type strain/MICΔohr strain)for OAOOH and LAOOH (more than 10-fold) was considerably greater than a similar parameter for artificial hydroperoxides (around threefold for t-BHP and CHP). For H2O2, the MIC was the same for the wild-type and Δohr strains (Table 1) and ChOOH did not inhibit the growth of either. These results indicated once again that fatty acid hydroperoxides are the best substrates for Ohr pro- teins, which correlates well with the original docking analysis (Fig. 1 and Fig. S1), the inactivation assay data (Fig. 3B and Fig. S4), and the second-order rate constant (k)values(Table1).Asimportant controls, the phenotypes of the Δohr strain were complemented in trans (pUCp18 plasmid) with the ohr gene from P. aeruginosa (LN04 strain) with the restoration of the wild-type resistance to hydroperoxides (Fig. 6). These controls unequivocally indicated that the observed sensitivities were due to the ohr gene deletion. Fur- thermore, nonoxidized lipids did not kill either wild-type or Δohr – strains (Fig. S7 A C), indicating that the hydroperoxide moiety in Fig. 6. Susceptibility of Δohr P. aeruginosa strain to various hydroperoxides. the carbon backbone was fundamental for inducing cell death. The MIC assay was performed to assess the sensitivities of P. aeruginosa strains The response of P. aeruginosa to fatty acid hydroperoxides was to distinct hydroperoxides: pUCp18 Ø is an empty vector, and pUCp18 ohr is a further investigated by analyzing other mutants to thiol- vector for expression of ohr from P. aeruginosa. The concentrations of hydro- dependent peroxidases. Remarkably, among all mutants, only Δohr peroxides used are described above each panel. Plates were incubated at 37 °C displayed sensitivity to LAOOH (Fig. 7 A and B), indicating the under agitation (200 rpm), and the results were observed after 16 h. MIC assays PaOhr is essential to the protection of P. aeruginosa to this insult. were done in biological triplicates, each one with two technical replicates. All Δ hydroperoxides were dissolved in DMSO, with the exception of ChOOH, which Furthermore, the ohr strain transformed with for other was dissolved in isopropyl alcohol. As control experiments, the same assays were thiol-dependent peroxidases did not recover the wild-type phe- performed with 5% DMSO or 5% isopropyl alcohol only or with the respective notype. For example, Gpx expression in the Δohr background fatty acid or cholesterol dissolved in DMSO and isopropyl alcohol, respectively, only partially restored the resistance to LAOOH treatment and no loss of viability were observed. Wt, wild type.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1619659114 Alegria et al. Downloaded by guest on September 30, 2021 Δohr strain was no more sensitive to ChOOH than the wild-type PNAS PLUS strain, indicating a lack of significant affinity between ChOOH and the enzyme. The AhpE/Ohr competitive assay developed here was straight- forward in comparison to other methods previously described, allowing us to obtain the second-order rate constants for the oxi- dation of peroxidatic Cys by distinct hydroperoxides. Importantly, we could use fatty acid hydroperoxides in the low micromolar range (1–2 μM), which is well below the CMC of long-chain fatty acid hydroperoxides (10–40 μM). With this approach (associated with the inactivation and the MIC assays), we could unambiguously establish that long-chain fatty acid hydroperoxides are more effi- ciently reduced by Ohr than artificial organic hydroperoxides and H2O2 (Table 1). In addition to fatty acid hydroperoxides, peroxynitrite was reduced − − Fig. 7. Response of P. aeruginosa to LAOOH and peroxynitrite. pUCp18 Ø is an with very high efficiency by XfOhr (on the order of 107 M 1·s 1). empty vector, and pUCp18 ohr, ahpC1, tpx,andgpx from P. aeruginosa are This finding was somehow unexpected because peroxynitrite is not a vectors for ohr, ahpC1, tpx,andgpx expression, respectively. (A and B)MIC assay to assess the sensitivities of P. aeruginosa strains deficient for Cys-based hydrophobic molecule, although the reaction of peroxynitrite with peroxidases to LAOOH. (C, Upper) Western blot for Ohr expression in P. aeru- thiols involves the protonated form of peroxynitrite (peroxynitrous ginosa using an antibody against Ohr from X. fastidiosa.(C, Lower) Ponceau S acid) and thiolates (48). It should be noted here that reactions was used as a loading control. Cells were exposed to SIN-1 (1 mM), DMSO (5%), of peroxynitrite with thiolates are much faster than similar reactions μ μ LAOOH (50 M), t-BHP (200 M), and MeOH (5%) for 30 min at 37 °C. WT, wild of thiolates with organic hydroperoxides or H2O2 (48–50). Un- type. (D) Colony count assay to assess the sensitivities of P. aeruginosa strains to fortunately, there is no report of the kinetics between fatty acid SIN-1 (3 mM). In all cases, cells were treated with ATZ (5 mM), a catalase in- hydroperoxides with noncatalytic thiols, such as free Cys or the single hibitor, 10 min before cells were challenged with SIN-1 (3 mM) for 30 min at reduced Cys residue in BSA. Nevertheless, using the reaction be- 37°C.Thebarsrepresentthemeansofthecolonyformationunit(c.f.u.)per- centage relative to the sensitivity of cells treated with DMSO (5%) + 3-ATZ tween t-BHP and ordinary thiolates as a reference (51), it is evident (5 mM) plus SD. The Δohr mutant was statistically more sensitive than the wild- that the gain in reactivity afforded by Ohr is two to three orders of BIOCHEMISTRY type strain (**P < 0.05, unpaired t test; n = 8). WT, wild type. magnitude for H2O2 and peroxynitrite, but six orders of magnitude for t-BHP (Table S1). According to the Brønsted relationship, the pKa of the leaving catalase inhibitor (Fig. S7 D–F). This observation is in agreement group is a factor that is involved in the reactivity of thiolates with with studies in E. coli, P. aeruginosa, and Francisella tularensis, hydroperoxides (13). The pKa values for aliphatic alkoxyl groups, where catalase enzymes are preponderant in the defense against which are the leaving groups corresponding to t-BHP and fatty exogenous H2O2 (44–47). acid hydroperoxides, are expected to have similar values (13). The response of P. aeruginosa to peroxynitrite is more diffi- Therefore, it is reasonable to think that the reactions of t-BHP cult to investigate, in part, due to the intrinsic instability of this and fatty acid hydroperoxides with ordinary thiols (e.g., the thiol hydroperoxide. In this case, we had to use a distinct assay, different of BSA) are within the same range and that the catalytic power from MIC, by exposing the bacterium to 3-morpholinosydnonimine of Ohr proteins for fatty acid hydroperoxides is therefore ex- hydrochloride (SIN-1, a peroxynitrite generator) in buffer and then traordinary (at least 106-fold increase). This high reactivity is assessing the number of viable colonies. A tendency of an increased probably related to its capacity to bind and orient the peroxyl sensitivity for Δohr cells in relation to the wild-type strain was de- group of fatty acid hydroperoxides toward the thiolate group of tected (Fig. S8D), which was statistically significant in cells, where Cys61 (Fig. 1), decreasing the free energy of the transition state. catalase was inhibited by ATZ (Fig. 7D). In contrast, both ATZ- In the case of the reduction of peroxynitrite by Ohr, the cor- treated and untreated wild-type cells were equally sensitive to responding rate constant is one of the highest determined so far SIN-1, and deletion of ahpC gene did not affect the viability of (52). In fact, the rate constant of peroxynitrite reduction by Ohr bacterial cells (Fig. S8C). Furthermore, the expression of ohr was is comparable to the rate constants reported for Prxs and thiol- induced in cells exposed to SIN-1 (Fig. 7C), implicating PaOhr in or selenol-dependent Gpxs (10, 12, 13, 53–55). Therefore, it is the response of P. aeruginosa to peroxynitrite. feasible that Ohr plays a relevant role in vivo in the reduction of peroxynitrite. This role might be significant, especially consid- Discussion ering that inducible nitric oxide synthase (and possibly perox- Previous work has indicated that substrate hydrophobicity is a ynitrite as a product of nitric oxide) displays a crucial role in the major physicochemical determinant of substrate specificity for pulmonary response to P. aeruginosa infection (42, 56). Also Ohr (16, 19, 21, 22). Accordingly, docking analysis performed here for plant pathogens (e.g., X. fastidiosa), peroxynitrite and other with XfOhr indicated that nonpolar contacts appear to be major nitric oxide-mediated processes are emerging as relevant players determinants in the binding of lipid hydroperoxides within the in defense responses (41). Accordingly, the levels of PaOhr are active site (Fig. 1 and Fig. S1A). The elongated shape of the sub- induced in cells exposed to the peroxynitrite generator (Fig. 7C), strate appears to be another feature to be considered presumably and the Δohr strain treated with ATZ is sensitive to this oxidant because the substrates, once docked, fill a Y-shaped cavity on the (Fig. 7D). However, at the moment, it is difficult to identify if the enzyme generating a large hydrophobic contact area, which would response of P. aeruginosa to SIN-1 is a direct effect of peroxynitrite be anticipated to contribute significantly to the interaction energy. or if it is due to some secondary derived oxidant. For instance, it is Indeed, cholesterol, which is highly hydrophobic but with a shape well known that lipids are targets for peroxynitrite oxidation and very distinct from PEG and fatty acids, did not dock well into the could release secondary products that might mount the adaptive XfOhr active site (Fig. S1B). Other biochemical (Figs. 2 and 3) and response of P. aeruginosa (57). The involvement of catalase in microbiological (Fig. 6) assays also indicated that ChOOH is not an the response of bacteria to peroxynitrite was shown previously in Ohr substrate. For example, in the case of the MIC assay (Fig. 6), F. tularensis and Salmonella enterica serovar Typhimurium,twomi- in addition to the natural surfactants of P. aeruginosa,Brij-58(1%) croorganisms devoid of Ohr (47, 58), although the latter does was supplemented during the experiments. Despite this fact, the contain an osmC gene. Our data indicate that especially Ohr and

Alegria et al. PNAS Early Edition | 7of10 Downloaded by guest on September 30, 2021 possibly catalase cooperate in the protection of P. aeruginosa ganic extraction was performed, and the products were analyzed by HPLC- against this oxidant (Fig. 7). tandem mass spectrometry. Mass spectrometry analysis was performed in a In addition to peroxynitrite, fatty acid hydroperoxides are in- triple-quadrupole instrument (Quattro II; Micromass) essentially as described volved in host–pathogen interactions (59, 60). Fatty acid hydro- by Miyamoto et al. (68). peroxides could be generated by nonenzymatic lipid peroxidation Analysis of Ohr Hyperoxidation and Inactivation. Freshly purified recombinant of cis-vaccenic acid and other monounsaturated fatty acids that XfOhr (300 μM) was reduced for 1 h using DTT (100 mM). Afterward, excessive are present in bacterial membranes, because most bacteria syn- DTT was removed by gel filtration (PD-10 desalting; GE Healthcare), and the −1 −1 thesize little or no polyunsaturated fatty acids (61). For this amount of protein recovered was quantified by its A280 (e = 3,960 M ·cm ; reason, it is assumed that nonenzymatic lipid peroxidation is not determined by the ProtParam tool ExPASy). Next, XfOhr (10 μM) was treated a favorable process in bacteria. Alternatively, Ohr may reduce with increasing amounts of hydroperoxides (as depicted in Fig. 3B and Fig. S4) polyunsaturated fatty acid hydroperoxides enzymatically gener- for 1 h at 37 °C in sodium phosphate buffer (20 mM), pH 7.4, containing DTPA ated by plant or animal hosts. In mammals, pathogens, such as (1 mM) and 0.15% (vol/vol) Triton X-100 or 0.25% (vol/vol) Tween-20. After these pretreatments, XfOhr was again submitted to gel filtration to P. aeruginosa, can activate host cytosolic PLA2 (62), releasing arachidonic acid (20:4 ratio) for oxidative modification by en- remove excessive hydroperoxides, and the recovered enzyme was again zymes, such as 5-lipoxygenase or 15-lipoxygenase (63). Products quantified by A280. Finally, the same sample of XfOhr was assayed by the lipoamide-lipoamide dehydrogenase–coupled assay (using t-BHP as a sub- of lipoxygenases have profound and distinct effects on the out- strate) and by nonreducing SDS/PAGE, which allow the assessment of the come of inflammation (59, 64). Given that endogenous levels of XfOhr oxidative state (22). fatty acid hydroperoxides, such as 5-hydroperoxyeicosatetraenoic

acid [5(S)-HpETE] or 15(S)-HpETE, regulate acute inflammation Digestion of Phosphatidylcholine Hydroperoxide with PLA2. This enzymatic (59), it is reasonable to hypothesize that Ohr might somehow im- digestion was performed as previously described (71). In a solution con-

pact the progression of the immune response. In plants, the im- taining CaCl2 (10 mM), KCl (35 mM), and Tris (20 mM) at pH 8.0, phospha- mune response is also mediated by oxidized products of fatty acids, tidylcholine (catalog no. P3556; Sigma) from egg yolk was added at a final derived from linoleic (18:2 ratio) and linolenic (18:3 ratio) acids in concentration equal to 100 μM. The solution was stirred for 1 min abruptly. – this case (60). Therefore, it is tempting to speculate that Ohr might Thereafter, PLA2 (P0861 Sigma Aldrich Co.) was added (final amount of subvert host immune pathways by some still unknown mechanisms 10 units), and the solution was then incubated for 15 min or 30 min at 40 °C under gentle agitation. After this time, the components of the dihydrolipoamide (65). Remarkably, P. aeruginosa contains endogenous enzymatic system and Triton X-100 were added for testing the activity of XfOhr on systems capable of oxidizing unsaturated fatty acids (especially phospholipids treated with PLA2. oleic acid) to hydroperoxide and hydroxide derivatives, whose bi- ological function is still elusive (66, 67). Moreover, the experi- Kinetics of Organic Hydroperoxide Reduction by Ohr. A competition method mental approach presented herein allowed dissecting the relevance was developed here, taking advantage of the fact that Ohr from X. fastid- of Ohr toward three different groups of biologically relevant hy- iosa and P. aeruginosa does not possess Trp residues, and consequently droperoxides, underscoring the complementary role that different displays low intrinsic fluorescence. In contrast, MtAhpE has intrinsic fluo- thiol- and heme-dependent peroxidases have on controlling cellu- rescence that decreases upon oxidation (8, 55). As expected, XfOhr practi- lar oxidative signaling and damage. cally lacks intrinsic fluorescence when excited at 295 nm, in contrast to MtAhpE (Fig. S5). Therefore, the assay developed here is based on the Materials and Methods competition between Ohr and MtAhpE for distinct hydroperoxides, moni- toring the intrinsic fluorescence of MtAhpE when exciting at 295 nm, either Reagents, Including Hydroperoxides. All reagents purchased had the highest following the time course of the reaction (using an SX20 Applied Photo- degree of purity. The t-BHP was obtained from Sigma–Aldrich Co. CHP and physics stopped-flow apparatus) or at final time points (using an Aminco H O were obtained Merck KGaA. XfOhr and PaOhr, as well as MtAhpE, 2 2 Bowman Series 2 luminescence spectrophotometer or a Cary Eclipse Fluo- were obtained and, in some experiments, reduced as previously described (8, rimeter). Rate constants for MtAhpE oxidation were determined for those 55). The 15(S)-HpETE and 5(S)-HpETE were purchased from Cayman Chemical. organic hydroperoxides that were not previously studied by Reyes et al. (8) and Hugo et al. (55). Photochemical Synthesis of Lipid Hydroperoxides. Oleic acid, linoleic acid, Concentrations of MtAhpE, Ohr, and hydroperoxide used were typically cholesterol, and phosphatidylcholine were photooxidized to the corre- 2 μM, 2–8 μM, and 1.8 μM, respectively. Because enzyme concentration de- sponding hydroperoxides according to the method of Miyamoto et al. (68). creased during the time course of the experiment, no pseudo–first-order This procedure is based on the oxidation of lipids by singlet molecular ox- conditions applied. Therefore, we used the following equation (Eq. 1) to find ygen produced in an O -saturated atmosphere using methylene blue as a 2 out the relationship between the rate constants for the reaction of a se- photosensitizer. OAOOHs, LAOOHs, and ChOOHs were purified by flash- lected hydroperoxide with Ohr (k ) and with MtAhpE (k ): column chromatography and were quantified by iodometry (68). The hy- OHR AhpE n o droperoxide isomers were not separated among themselves, only from the ½ C. Prot ln red 0 k ½ C. Prot − ½ C. Prot∞ parental lipids. Phosphatidylcholine hydroperoxide was purified by HPLC C. Prot = nred 0 ox o ½ OHR , [1] kOHR red 0 and was quantified by phosphorus assay and iodometry (69). ln ½ − ½ red OHR 0 ox OHR ∞

Docking Lipid Hydroperoxides in Ohr. Using the X. fastidiosa Ohr structure where kC. Prot is the rate constant for the reaction between a selected hydro- (PDB ID code 1ZB8) as a template, docking analysis was carried out using peroxide with the protein competing with Ohr for it (in this case, reduced Gold software, version 5.0 (70), utilizing organic hydroperoxides with dis- MtAhpE, whose reactivity with the hydroperoxide is either already known or

tinct chemical structures. determined herein, as described in Fig. S6); kOHR is the rate constant of the reaction between the hydroperoxide with reduced Ohr; [redC. Prot]0 is the initial Lipoamide-Lipoamide Dehydrogenase Peroxidase-Coupled Assay. The lipoyl concentration of the reduced competing protein; [oxC. Prot]∞ corresponds to peroxidase activity of Ohr was determined as previously described (23). Re- the final concentration of the competing protein in the oxidized state; [redOhr]0 −1 −1 actions were followed by decay of A340 (e = 6,290 M ·cm ) due to NADH is the initial concentration of reduced Ohr; and [oxOhr]∞ is the final concen- oxidation. In some cases, several hydrophobic hydroperoxides were solubi- tration of oxidized Ohr, determined as [oxC. Prot]∞ formed in the absence minus lized in Tween-20 or Triton X-100 as described throughout the main text and the presence of Ohr (50). The final concentration of MtAhpE in the oxidized appropriate controls were performed. state was determined assuming an equimolar reaction with hydroperoxide in the absence of the competing protein. Mass Spectrometry Analysis of the Products Formed in the Ohr Reaction with LAOOH and OAOOH. OAOOH or LAOOH (4 μM) was incubated in the presence Kinetics of Peroxynitrite Reduction by Ohr. The reduction of peroxynitrite by of an equimolar concentration of reduced XfOhr for 1 min at 37 °C in sodium XfOhr was investigated using different methodologies. First, the initial rate phosphate buffer (20 mM), pH 7.4, containing diethylenetriaminepenta- of peroxynitrite decomposition was followed at 310 nm due to its intrinsic acetic acid (DTPA) (0.1 mM). As a control reaction, OAOOH and LAOOH were absorbance in the absence and presence of increasing concentrations of incubated with previously alkylated Ohr (0.2 mM NEM). Afterward, an or- reduced XfOhr in 100 mM (pH 7.4) sodium phosphate buffer containing

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1619659114 Alegria et al. Downloaded by guest on September 30, 2021 DTPA (0.1 mM) at 25 °C, using an SX20 Applied Photophysics stopped-flow supplemented with 1% Brij-58. Concentrations of hydroperoxides and con- PNAS PLUS apparatus (50). In a second approach, the kinetics of peroxynitrite reduction ditions of solubilization are described in Figs. 6 and 7 and Fig. S7. were indirectly determined using a competition assay between reduced XfOhr and HRP for peroxynitrite (10, 11, 50). HRP compound I formation was mea- Colony Counting Assay to Assess SIN-1 Sensitivity. Cultures of P. aeruginosa

sured at various concentrations of reduced XfOhr, and experimental curves were grown on LB medium at 37 °C until OD600 = 1.Afterthatpoint, were fitted to exponential curves before the slow decay of compounds I –II cultures were washed twice in Dulbecco PBS (Life Technologies) and di- – occurred. Because no pseudo first-order conditions applied (reduced XfOhr luted to OD600 = 0.01 in the same buffer. A volume of 200 μLofthecell concentration decreased during the time course of the experiment), we used suspension was incubated with 5 mM catalase inhibitor ATZ for 10 min at Eq. 1 to find out the relationship between the kHRP (the C. Prot in this assay) room temperature without agitation. After that time, 6.2 μLof96mM and kOHR. SIN-1 solution (diluted in DMSO) was added at the bottom of the tube and The final concentration of compound I was quantitated using the reported incubated for 30 min at 37 °C at 200 rpm, and treated cultures were then e = −1· −1 398 nm 42,000 M cm and the final concentration of oxidized XfOhr, serially diluted in 10 mM MgSO4 and plated on solid LB medium to count determined as compound I formed in the absence minus the presence of the number of viable cells by colony formation unit. As a control, the same XfOhr (11). To perform these calculations, we previously determined the treatment was carried out only with DMSO. The results were expressed by 6 −1 −1 rate constant between HRP and peroxynitrite as 2.2 × 10 M ·s at pH 7.4 the relative percentage, compared with controls, of viable cells remaining and 25 °C for the specific batch and experimental conditions used in the after treatment. The statistical analysis was performed using an unpaired experiments described here. Indeed, the rate constant changed from batch t test (P < 0.05). to batch of HRP (72). Finally, the competition approach between reduced Ohr and MtAhpE Ohr Expression upon Oxidative Insults. Cultures of P. aeruginosa (PA14, Δohr, described above was also used for determining the kinetics of peroxynitrite- and ΔohrR) were grown on M9 medium supplemented with 0.2% glucose at mediated MtAhpE oxidation. 37 °C and 200 rpm until OD600 = 0.5. After that time, 500 μL of each culture was separated in different flasks to receive the treatments (200 μM t-BHP, Strains and Growth Conditions. P. aeruginosa UCBPP-PA14 (73) wild-type and 50 μM LAOOH, and 1 mM SIN-1). As a control, the same volumes of the MAR2xT7 mutants (74) (see Table S2), and E. coli strains were grown in solvents in which the previous compounds were diluted [water, 5% (vol/vol) lysogenic broth (LB) medium at 37 °C supplemented, when necessary, with methanol, and 5% (vol/vol) DMSO, respectively] were added. After 30 min of μ μ μ gentamicin (10 g/mL), carbenicillin (300 g/mL), or ampicillin (100 g/mL). treatment (37 °C, 200 rpm), cells were pelleted and then resuspended in Laemmli buffer and processed for SDS/PAGE analysis. The gel was electro- Cloning for Complementation Assays in P. aeruginosa. The coding region from ohr blotted onto a 0.45-μm nitrocellulose membrane in Tris·glycine buffer at 24 gene (PA14_27220) with its 150-bp upstream sequence and ahpC (PA14_01710), V/600 mA for 30 min. Blotted membranes were blocked for 3 h at room ahpC2 (PA14_18690), tpx (PA14_31810), and gpx (PA14_27520) genes (corre- temperature in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) sponding to fragments of 780 bp, 561 bp, 600 bp, 485 bp, and 490 bp, re- and 5% (vol/wt) nonfat dry milk (Bio-Rad). The membrane was then in- BIOCHEMISTRY spectively) were amplified using oligonucleotides described in Table S3). With cubated with a rabbit anti-XfOhr polyclonal serum (1:1,000 in TBS-T) over- the exception of ohr, all genes were cloned without a stop codon and har- night at 4 °C, followed by washing with TBS-T. The membrane was boring a human influenza hemagglutinin (HA) tag inserted at 3′, allowing us to subsequently incubated with an anti-rabbit IgG-alkaline phosphatase anti- check their expression levels in P. aeruginosa cells by Western blotting. body (KPL, Inc.; 1:1,000 in TBS-T) for 2 h. After washing, the proteins were The final products were cloned into EcoRI and BamHI sites of the broad- detected following incubation with alkaline phosphatase substrates: 5-bromo- host range vector pUCp18 (GenBank accession no. U07164) previously 4-chloro-3-indolyl phosphate (0.3 mg/mL)/nitro blue tetrazolium (0.1 mg/mL) digested with the same enzymes. The pUCp18 constructs were introduced on an appropriate buffer [10 mM Tris·HCl (pH 9.0), 100 mM NaCl, and into P. aeruginosa PA14 wild-type and MAR2xT7 transposon ohr mutant 5 mM MgCl2]. (Δohr) cells by electroporation (75). As a control, the same strains were transformed with empty pUCp18 vector. All PCR reactions were carried out ACKNOWLEDGMENTS. We thank Dr. Regina Baldini [Instituto Quimica, Uni- with Thermo Scientific High Fidelity Taq Polymerase using genomic DNA of versidade de São Paulo (USP)] and Laurence G. Rahme (Harvard University) for PA14 as a template. Errors from PCR reactions were checked by sequencing providing PA14 and strains from a transposon library. We also thank Adriano de the clones. Britto Chaves-Filho (Instituto Quimica, USP) for his support in mass spectrometry analysis. This work was financially supported by Fundaca̧odeAmparoã ̀Pes- ̃ MIC Assay. Cultures of P. aeruginosa (PA14 and Δohr) were grown on LB quisa do Estado de Sao Paulo (FAPESP) Grant 13/07937-8 (Redox Processes medium at 37 °C until OD = 1. After that time, cultures were diluted in in Biomedicine) (to M.A.d.O., S.M., P.D.M., O.A., and L.E.S.N.), NIH Grant 600 1R01AI095173 (to R.R.), a grant from the Universidad de la República (Comisión fresh LB medium supplemented with an appropriate antibiotic to OD = 600 Sectorial de Investigación Científica, Uruguay; to R.R.), and a grant from μ 0.01; then, 150 L was dispensed in 96-well plates previously loaded with Ridaline (Fundación Manuel Pérez) (to R.R.). M.H. was the recipient of a fellowship different concentrations of tested hydroperoxides. Afterward, assay plates from the Agencia Nacional de Investigación e Innovación (Uruguay), and T.G.P.A. were incubated at 37 °C under agitation (200 rpm), and the results were (2008/07971-3) and D.A.M. (2012/21722-1) were recipients of fellowships from recorded after 16 h. To increase the solubility of ChOOH, LB medium was FAPESP.

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